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MOLECULAR STRUCTURE AND BIOLOGIC ACTIVITY Chemical compounds, usually derived from plants and other natural sources, have been used by humans for thousands of years to alleviate pain, diarrhea, infection, and various other maladies. Until the 19th century, these “remedies” were primarily crude preparations of plant material of unknown constitution. The revolution in synthetic organic chemistry during the 19th century produced a concerted effort toward identiﬁcation of the structures of the active constituents of these naturally derived medicinals and synthesis of what were hoped to be more efﬁcacious agents. By determining the molecular structures of the active components of these complex mixtures, it was thought that a better understanding of how these components worked could be elucidated.

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RELATIONSHIP BETWEEN MOLECULAR STRUCTURE AND BIOLOGIC ACTIVITY Molecular structure inﬂuences the biologic activity of chemical entities and that alterations in structure produce changes in biologic action. The structure of a molecule, its composition and arrangement of functional groups, determines the type of pharmacologic effect that it possesses (i.e., SAR). SARs are the underlying principle of medicinal chemistry. Similar molecules exert similar biologic actions in a qualitative sense. A corollary to this is that structural elements (functional groups) within a molecule most often contribute in an additive manner to the physicochemical properties of a molecule and, therefore, to its biologic action. Overall, structural organization also determines selectivity in activity.

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In the quest for better medicinal agents (drugs), it must be determined which functional groups within a speciﬁc structure are important for its pharmacologic activity and how these groups can be modiﬁed to produce more potent, more selective, and safer compounds. In order for drug molecules to exhibit their pharmacologic activity, they must interact with a biologic target, typically an enzyme, nucleic acid, or excitable membrane or other biopolymer (RECEPTOR CONCEPT). These interactions occur between the functional groups found in the drug molecule and those found within each biologic target. RELATIONSHIP BETWEEN MOLECULAR STRUCTURE AND BIOLOGIC ACTIVITY

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PHYSICOCHEMICAL PROPERTIES OF DRUGS Acid–Base Properties The human body is 70 to 75% water, which amounts to approximately 51 to 55 L of water for a 73-kg individual. For an average drug molecule with a molecular weight of 200 g/mol and a dose of 20 mg, this leads to a solution concentration of approximately 2 × 10−6 M (2 mM). When considering the solution behavior of a drug within the body, we are dealing with a dilute solution, for which the Brönsted- Lowry (8) acid–base theory is most appropriate to explain and predict acid–base behavior. This is a very important concept in medicinal chemistry, because the acid–base properties of drug molecules have a direct effect on absorption, excretion, and compatibility with other drugs in solution.

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Remember Bronsted Lowry acid-base concept When an acidic functional group loses its proton (often referred to as having undergone “dissociation”), it is left with an extra electron and becomes negatively charged. This is the “ionized” form of the acid. When a basic functional group is converted to the corresponding conjugate acid, it too becomes ionized. In this instance, however, the functional group becomes positively charged due to the extra proton. Functional groups that cannot give up or accept a proton are considered to be “neutral”

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An example Chemical structure of ciprofloxacin (i.e., a ﬂuoroquinolone antibacterial agent) showing the various organic functional groups. Depending on the pH of the physiologic environment, this molecule will either accept a proton (secondary alkylamine), donate a proton (carboxylic acid), or both. Thus, it is described as amphoteric (both acidic and basic) in nature.

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Common Organic Functional Groups That are Considered Neutral Under Physiologic Conditions

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Predicting the Degree of Ionization of a Molecule: The Henderson-Hasselbalch equation for calculating percent ionization By knowing if there are acidic and/or basic functional groups present in a molecule, one can predict whether a molecule is going to be predominantly ionized or un-ionized at a given pH. The Henderson-Hasselbalch equation (Eq. 2.6) can be used to calculate the percent ionization of a compound at a given pH.

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An example

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Water Solubility of Drugs The solubility of a drug molecule in water greatly affects the routes of administration that are available, as well as its absorption, distribution, and elimination. Two key concepts to keep in mind when considering the water (or fat) solubility of a molecule are the potential for hydrogen bond formation and ionization of one or more functional groups within the molecule.

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Hydrogen Bonds Each functional group capable of donating or accepting a hydrogen bond contributes to the overall water solubility of the compound and increases the hydrophilic (water-loving) nature of the molecule. Conversely, functional groups that cannot form hydrogen bonds do not enhance hydrophilicity and will contribute to the hydrophobic (water-fearing) nature of the molecule. Hydrogen bonds are a special case of what are usually referred to as dipole–dipole interactions. A permanent dipole occurs with each of these atoms along a single bond (one atom has a partial negative charge, and one atom has a partial positive charge). The atom with a partial negative charge has higher electron density than the other atom. When the positive end of the dipole is a hydrogen atom, this interaction is referred to as a “hydrogen bond” (or H-bond).

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Even though the energy associated with each hydrogen bond is small (1 to 10 kcal/mol/bond), it is the additive nature of multiple hydrogen bonds that contributes to the overall water solubility of a given drug molecule. This type of interaction is also important in the interaction between a drug and its biologic target (e.g., receptor). Hydrogen Bonds An example of hydrogen bonding between water and hypothetical drug molecules.

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The importance of ionization on water solubility In addition to the hydrogen-bonding capacity of a molecule, another type of interaction plays an important role in determining water solubility: the ion–dipole interaction. This type of interaction can occur with organic salts. Ion–dipole interactions occur between either a cation and the partially negatively charged atom found in a permanent dipole (e.g., the oxygen atom in water) or an anion and the partially positively charged atom found in a permanent dipole (e.g., the hydrogen atoms in water). Examples of ion–dipole interactions.

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Drugs and their salt forms Organic salts are composed of a drug molecule in its ionized form and an oppositely charged counterion. For example, the salt of a carboxylic acid is composed of the carboxylate anion (ionized form of the functional group) and a positively charged ion (e.g., Na+) and the salt of a secondary amine is composed of the ammonium cation (ionized form of the functional group and a negatively charged ion; e.g., Cl−). Not all organic salts are very water soluble. To associate with enough water molecules to become soluble, the salt must be highly dissociable; in other words, the cation and anion must be able to separate and interact independently with water molecules. Highly dissociable salts are those formed from strong acids with strong bases (e.g., sodium chloride), weak acids with strong bases (e.g., sodium phenobarbital), or strong acids with weak bases (e.g., atropine sulfate).

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Examples Water solubilities of different salt forms of selected drugs.

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Another example Because of the presence of three very polar functional groups (two of them being ionizable), one would expect tyrosine to be very soluble in water, yet its solubility is only 0.45 g/1,000 mL. The basic alkylamine (pKa 9.1 for the conjugate acid) and the carboxylic acid (pKa 2.2) are both ionized at physiologic pH, and a zwitterionic molecule results. These two charged groups are sufﬁciently close that a strong ion–ion interaction occurs, thereby keeping each group from participating in ion– dipole interactions with surrounding water molecules. This lack of interaction between the ions and the dipoles found in water results in a molecule that is very water insoluble

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FACTORS ACTING ON THE BIOLOGICAL ACTIVITY OF A DRUG MOLECULE STEREOCHEMISTRY AND DRUG ACTION Stereoisomers are molecules that contain the same number and kinds of atoms, the same arrangement of bonds, but different three-dimensional structures; in other words, they only differ in the three-dimensional arrangement of atoms in space. There are two types of stereoisomers: enantiomers and diastereomers. Enantiomers are pairs of molecules for which the three-dimensional arrangement of atoms represents nonsuperimposable mirror images. Diastereomers represent all of the other stereoisomeric compounds that are not enantiomers. Thus, the term “diastereomer” includes compounds that contain double bonds (geometric isomers) and ring systems. Unlike enantiomers, diastereomers exhibit different physicochemical properties, including, but not limited to, melting point, boiling point, solubility, and chromatographic behavior. These differences in physicochemical properties allow the separation of individual diastereomers from mixtures with the use of standard chemical separation techniques, such as column chromatography or crystallization.

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FACTORS ACTING ON THE BIOLOGICAL ACTIVITY OF A DRUG MOLECULE STEREOCHEMISTRY AND DRUG ACTION Enantiomers cannot be separated using such techniques unless a chiral environ-ment is provided or if they are ﬁrst converted to diastereomers (e.g., salt formation with another enantiomer). The physicochemical properties of a drug molecule are dependent not only on what functional groups are present in the molecule but also on the spatial arrangement of these groups. This becomes an especially important factor when the environment that a molecule is in is asymmetric, such as the human body. Proteins and other biologic targets are asymmetric in nature. How a particular drug molecule interacts with these macromolecules is determined by the three-dimensional orientation of the functional groups present. If critical functional groups in the drug molecule do not occupy the proper spatial region, then productive interactions with the biologic target will not be possible. As a result, it is possible that the desired pharmacologic activity will not be achieved. If, however, the functional groups within a drug molecule are located in the proper three-dimensional orientation, then the drug can participate in multiple key interactions with its biologic target.

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FACTORS ACTING ON THE BIOLOGICAL ACTIVITY OF A DRUG MOLECULE STEREOCHEMISTRY AND DRUG ACTION Approximately one in every four drugs currently on the market is some type of isomeric mixture. For many of these drugs, the biologic activity may only reside in one isomer (or at least predominate in one isomer). The majority of these isomeric mixtures are termed “racemic mixtures” (or “racemates”). A racemic mixture is comprised of equal amounts of both possible drug enantiomers. When enantiomers are introduced into an asymmetric, or chiral, environment, such as the human body, they display different physicochemical properties. This can lead to signiﬁcant differences in their pharmacokinetic and pharmacodynamic behavior, resulting in adverse side effects or toxicity. For example, the individual isomers in a racemic mixture can exhibit signiﬁcant differences in absorption (especially active transport), serum protein binding, and metabolism. As it relates to drug metabolism, it is certainly possible that only one of the isomers can be converted into a toxic substance or can inﬂuence the metabolism of another drug.

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FACTORS ACTING ON THE BIOLOGICAL ACTIVITY OF A DRUG MOLECULE STEREOCHEMISTRY AND DRUG ACTION

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Stereochemistry and Biologic Activity Easson-Stedman Hypothesis In 1933, Easson and Stedman reasoned that differences in biologic activity between enantiomers resulted from selective reactivity of one enantiomer with its receptor. They postulated that such interactions require a minimum of a three-point ﬁt to the receptor. Optical isomers. Only in compound 6 do the functional groups A, B, and C align with the corresponding sites of binding on the asymmetric surface.

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An example: The differences in vasopressor activity of R-(−)- epinephrine, S-(+)-epinephrine, and the achiral N-methyldopamine With R-(−)-epinephrine, the three points of interaction with the receptor site are the substituted aromatic ring, b-hydroxyl group, and the protonated secondary ammonium group. All three functional groups interact with their complementary sites on the receptor surface, resulting in receptor stimulation (in this case). With S-(+)-epinephrine, only two interactions are possible (the protonated secondary ammonium and the substituted aromatic ring). The b-hydroxyl group is located in the wrong place in space and, therefore, cannot interact properly with the receptor. N-methyldopamine can achieve the same interactions with the receptor as S-(+)-epinephrine; therefore, it is not surprising that its vasopressor response is the same as that of S-(+)-epinephrine and less than that of R-(−)-epinephrine.

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Homework Determine the chiral centers in the molecules below, and draw each stereoisomer.

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Geometric isomers Restricted bond rotation caused by carbon–carbon double bonds (alkenes or oleﬁns) and similar systems, such as imines (C =N), can produce stereoisomers. These are also referred to as geometric isomers, although they more properly are classiﬁed as diastereomers. In this situation, substituents can be oriented on the same side or on opposite sides of the double bond.

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Conformational Isomerism Conformational isomerism takes place via rotation about one or more single bonds. Such bond rotation results in nonidentical spatial arrangement of atoms in a molecule. This type of isomerism does not require much energy because no bonds are broken. In the conversion of one enantiomer into another (or diastereomer) bonds are broken, which requires signiﬁcantly more energy. The neurotransmitter acetylcholine can be used to demonstrate the concept of conformational isomers.

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DRUG DESIGN: DISCOVERY AND STRUCTURAL MODIFICATION OF LEAD COMPOUNDS The process of drug discovery begins with the identiﬁcation of new, previously undiscovered, biologically active compounds, often called “hits,” which are typically found by screening many compounds for the desired biologic properties. We will next explore the various approaches used to identify “hits” and to convert these “hits” into “lead” compounds and, subsequently, into drug candidates suitable for clinical trials. Sources of “hits” can originate from natural sources, such as plants, animals, or fungi; from synthetic chemical libraries, such as those created through combinatorial chemistry or historic chemical compound collections; from chemical and biologic intuition due to years of chemical–biologic training; from targeted/rational drug design; or from computational modeling of a target site such as an enzyme. Chemical or functional group modiﬁcations of the “hits” are performed in order to improve the pharmacologic, toxicologic, physiochemical, and pharmacokinetic properties of a “hit” compound into a “lead” compound.

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DRUG DESIGN: DISCOVERY AND STRUCTURAL MODIFICATION OF LEAD COMPOUNDS The lead compound to be optimized should be of a known chemical structure and possess a known mechanism of action, including knowledge of its functional groups (pharmacophoric groups) that are recognized by the receptor/active site and are responsible for that molecule’s afﬁnity at the targeted receptor site. “Lead optimization” is the process whereby modiﬁcations of the functional groups of the lead compound are carried out in order to improve its recognition, afﬁnity, and binding geometries of the pharmacophoric groups for the targeted site (a receptor or enzyme); its pharmacokinetics; or its reactivity and stability toward metabolic degradation. The ﬁnal step of the drug discovery process involves rendering the lead compound into a drug candidate that is safe and suitable for use in human clinical trials, including the preparation of a suitable drug formulation.

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Natural Product Screening Following “leads” from folklore medicine, chemists of the late 19th and early 20th centuries began to seek new medicinals from plant sources and to assay them for many types of pharmacologic actions. This approach to drug discovery is often referred to as “natural product screening.” Before the mid-1970s, this was one of the major approaches to obtaining new chemical entities as “leads” for new drugs. Unfortunately, this approach fell out of favor and was replaced with the rational approaches to drug design developed during that period.

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Natural Product Screening

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Drug Discovery via Random Screening of Synthetic Organic Compounds The random screening of synthetic organic compounds approach to the discovery of new chemical entities for a particular biologic action began in the 1930s, after the discovery of the sulfonamide class of antibacterials. This random screening approach was also applied in the 1960s and 1970s in an effort to ﬁnd agents that were effective against cancer. Some groups did not limit their assays to identify a particular type of biologic activity but, rather, tested compounds in a wide variety of assays. This large-scale screening approach of drug “leads” is referred to as high-throughput screening, which involves the simultaneous bioassay of thousands of compounds in hundreds to thousands of bioassays.

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Drug Discovery from Targeted Dedicated Screening and Rational Drug Design Rational drug design is a more focused approach that uses greater knowledge (structural information) about the drug receptor (targets) or one of its natural ligands as a basis to design, identify, or create drug “leads.” Testing is usually done with one or two models (e.g., speciﬁc receptor systems or enzymes) based on the therapeutic target. The drug design component often involves molecular modeling and the use of quantitative structure–activity relationships (QSARs) to better deﬁne the physicochemical properties and the pharmacophoric groups that are essential for biologic activity. The development of QSARs relies on the ability to examine multiple relationships between physical properties and biologic activities. This approach needs evaluation of the nature of interaction forces between a drug and its biological target, as well as the ability to predict activity in molecules. The methodology is better for the development of a lead compound into a drug candidate than for the discovery of a lead compound.

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An example: Current Medicinal Chemistry, 2000, 7,

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Drug Discovery via Drug Metabolism Studies Metabolites of known drug entities are isolated and assayed for biologic activity using either the same target system or broader screen target systems. The broader screening systems are more useful if the metabolite under evaluation is a chemical structure that was radically altered from the parent molecule through some unusual metabolic rearrangement reaction. In most cases, the metabolite is not radically different from the parent molecule and, therefore, would be expected to exhibit similar pharmacologic effects. One advantage of evaluating this type of drug candidate is that a metabolite can possess better pharmacokinetic properties, such as a longer duration of action, better oral absorption, or less toxicity with fewer side effects.

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An example

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Drug Discovery from the Observation of Side Effects Phase 4 studies are referred to as pharmacoviligance nowadays. The gaining of information not only aids to observe patients particularly for side effects following the launch of the drug to the market but also to design new molecules to overcome the side effects. Sometimes side effects might be quite important to identify and validate a new pharmacological target taht would result in the generation of new compounds. Minoxidil was first discovered and generated for antihypertensive purpose, nowadays it is used to prevent hair loss though.

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Another example Lots sulfonamide drugs were designed and synthesized at the first half of 20th century for the treatment of bacterial infections. Two examples are below: The side effect common with these compounds was alkaline diuresis. Later, it was realized that due to carbonic anhydrase inhibition. Even though these compounds have no use for antibacterial activity, the mechanism of side effect lead to the generation of sulfonamide compouds used as diuretics in hypertension treatments either alone or in combination therapy.

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Refinement of the Lead Structure Determination of the Pharmacophore Once a “hit” compound has been discovered for a particular therapeutic use, the next step is to identify the pharmacophoric groups. The pharmacophore of a drug molecule is that portion of the molecule that contains the essential functional group(s) that directly bind with the active site of the biologic target to produce the desired biologic activity. Because drug–target interactions can be very speciﬁc (think of a lock [receptor] and key [drug] relationship), the pharmacophore can constitute a small portion of the molecule.

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Another example: Morphine pharmacophore and its relationship to analgesic derivatives.

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Note: Modification to determine pharmacophore groups or hit molecules can result in drastic changes. Example: Effect of alkyl chain length on activity of morphine.

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Functional Group Modification: Bioisosterism When a lead compound is ﬁrst discovered, it often lacks the required potency and pharmacokinetic properties suitable for making it a viable clinical candidate. These can include undesirable side effects, physicochemical properties, other factors that affect oral bioavailability and adverse metabolic or excretion properties. These undesirable properties are often the result of the presence of speciﬁc pharmacophoric (functional) groups in the molecule. Successful modiﬁcation of the compound to reduce or eliminate these undesirable features without losing the desired biologic activity is the goal. Replacement or modiﬁcation of speciﬁc phar-macophoric (functional) groups with other groups having similar properties is known as “isosteric replacement” or “bioisosteric replacement.”

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Functional Group Modification: Bioisosterism Classical Bioisosteres (Groups Within the Row Can Replace Each Other)

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Example: Isosteric replacement of OH by NH 2 in folic acid and possible tautomers of folic acid and aminopterin.